Methods of treating cancer with a P2X7 peptide

ABSTRACT

The invention relates to purinergic (P2X) receptors, more specifically to P2X7 receptors, the generation of antibodies and the use of antibodies and immunogens that are capable of selectively binding to a non ATP-binding P2X7 receptor but not to an ATP-binding P2X7 receptor, for the detection and treatment of disease conditions, especially cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/877,715 filed Oct. 7, 2015, which is a continuation of U.S.application Ser. No. 13/841,692 filed Mar. 15, 2013, which is adivisional of U.S. application Ser. No. 12/677,799 filed Jul. 1, 2010,which is the US national stage of PCT/AU2008/001364 filed Sep. 12, 2008,now U.S. Pat. No. 8,440,186, the contents of each of which areincorporated by reference in their entirety. PCT/AU2008/001364 claimspriority to Australian application no. 2007905018 filed Sep. 14, 2007.

REFERENCE TO A SEQUENCE LISTING

This application includes an electronic sequence listing in a file named510348_SEQLST.txt, created on Mar. 1, 2018, and containing 28,485 bytes,which is incorporated by reference.

FIELD OF THE INVENTION

The invention relates to purinergic (P2X) receptors, to generation ofantibodies and to use of antibodies and immunogens for detection andtreatment of a disease or condition, especially cancer.

BACKGROUND OF THE INVENTION

Purinergic (P2X) receptors are ATP-gated cation-selective channels. Eachreceptor is made up of three protein subunits or monomers. To date sevenseparate genes encoding P2X monomers have been identified: P2X1, P2X2,P2X3, P2X4, P2X5, P2X6, P2X7.

P2X7 receptors are of particular interest as the expression of thesereceptors is understood to be limited to cells having potential toundergo programmed cell death, such as thymocytes, dendritic cells,lymphocytes, macrophages and monocytes. There is some expression of P2X7receptors in normal homeostasis, such as on erythrocytes.

Interestingly, a P2X7 receptor containing one or more monomers having acis isomerisation at Pro210 (according to SEQ ID NO: 1 in FIG. 1) andwhich is devoid of ATP binding function has been found on cells that areunderstood to be unable to undergo programmed cell death, such aspreneoplastic cells and neoplastic cells.

Antibodies generated from immunisation with a peptide including Pro210in cis bind to P2X7 receptors that are devoid of ATP binding function.However, they do not bind to P2X7 receptors capable of binding ATP.Accordingly, these antibodies are useful for selectively detecting manyforms of carcinoma and haemopoietic cancers and to treatment of some ofthese conditions.

The region of the P2X7 receptor containing Pro210 forms part of theextra cellular domain of the receptor. Antibodies raised against otherepitopes on this domain, including those that bind regions includingfrom Val71 to Val87 (according to SEQ ID NO: 1 in FIG. 1) and from Lys137 to Cys152 (according to SEQ ID NO:1 in FIG. 1) have been found notto be capable of selectively binding to the receptor that is devoid ofATP binding function. Hence, other than the antibodies directed to theregion including Pro210, no other antibodies have been found to date tobe able to discriminate between ATP and non ATP binding receptors.

There is a need for reagents for detection of cancer and in thiscontext, for new antibodies capable of discriminating between ATP andnon ATP binding P2X7 receptors. There is also a need for new cancertherapeutics, including antibodies and immunogens for providing an antitumour response.

SUMMARY OF THE INVENTION

In certain embodiments there is provided a peptide:

-   -   consisting of the sequence of SEQ ID NO: 2;    -   consisting of a sequence within the sequence of SEQ ID NO: 2,        said peptide being useful as an immunogen to generate an        antibody that is capable of selectively binding to a non        ATP-binding P2X7 receptor but not to an ATP-binding P2X7        receptor;    -   consisting of a sequence of SEQ ID NO: 3, said peptide being        useful as an immunogen to generate an antibody that is capable        of selectively binding to a non ATP-binding P2X7 receptor but        not to an ATP-binding P2X7 receptor; or    -   consisting of a sequence within the sequence of SEQ ID NO: 3,        said peptide being useful as an immunogen to generate an        antibody that is capable of selectively binding to a non        ATP-binding P2X7 receptor but not to an ATP-binding P2X7        receptor.

In other embodiments there is provided an antibody or fragment thereof:

-   -   capable of binding to a peptide described above; or    -   capable of binding to an epitope that includes one or more        residues of a peptide having a sequence of SEQ ID NO: 2.

In other embodiments there is provided an immune complex formed from thebinding of an antibody or fragment thereof described above to a nonATP-binding P2X7 receptor, monomer or fragment thereof, or to a peptideas described above.

In certain embodiments there is provided a method for determiningwhether a cell, tissue or extra cellular body fluid includes a nonATP-binding P2X7 receptor, monomer or fragment thereof including:

-   -   contacting a cell, tissue or extra cellular body fluid with an        antibody or fragment thereof in conditions for forming an immune        complex as described above, and    -   detecting whether an immune complex has been formed,

wherein detection of an immune complex determines that a cell, tissue orextra-cellular body fluid includes a non ATP-binding P2X7 receptor,monomer or fragment thereof.

In other embodiments there is provided a method for determining whethera cell, tissue or extra-cellular body fluid contains an antibody againsta non ATP-binding P2X7 receptor, monomer or fragment thereof including:

-   -   contacting a cell, a tissue or an extra-cellular body fluid with        a peptide as described above in conditions for forming an immune        complex between the peptide and an antibody in the cell, tissue        or extra-cellular body fluid, and    -   detecting whether an immune complex has been formed,

wherein detection of an immune complex determines that a cell, tissue orextra-cellular body fluid contains an antibody against a non ATP-bindingP2X7 receptor, monomer or fragment thereof.

In yet further embodiments there is provided a kit or composition fordetermining whether a cell, tissue or extra-cellular body fluid containsa non ATP-binding P2X7 receptor, monomer or fragment thereof, or anantibody against same including:

-   -   a peptide as described above; and/or    -   an antibody or fragment thereof as described above; and/or    -   non ATP-binding P2X7 receptor, monomer or fragment thereof; and        optionally    -   a further antibody for binding to the peptide, antibody or        fragment thereof or the non ATP-binding P2X7 receptor, monomer        or fragment thereof;    -   written instructions for use of the kit in a method described        above.

In other embodiments there is provided a pharmaceutical compositionincluding an antibody or fragment thereof as described above, or apeptide as described above together with a pharmaceutically acceptablecarrier, diluent or excipient.

In related embodiments there is provided a method of treatment of adisease characterised by the expression of a non ATP-binding P2X7receptor, monomer or fragment thereof including the step of providing anantibody or fragment thereof as described above, or a peptide asdescribed above to an individual requiring said treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows SEQ ID NO:1.

FIG. 2 shows SEQ ID NO:2

FIG. 3 shows SEQ ID NO:3

FIG. 4 shows SEQ ID NO:4

FIG. 5 shows SEQ ID NO:5

FIG. 6 shows SEQ ID NO:6

FIG. 7 shows SEQ ID NO:7.

FIG. 8 shows SEQ ID NO:8

FIG. 9 shows SEQ ID NO:9

FIG. 10 shows SEQ ID NO:10

FIG. 11 shows SEQ ID NO:11

FIG. 12 shows SEQ ID NO:12

DETAILED DESCRIPTION OF THE EMBODIMENTS

For the following descriptions, the technical and scientific terms usedherein will have the meanings commonly understood by one of ordinaryskill in the art, unless specifically defined otherwise.

The inventors have identified an epitope that is exclusively expressedon non ATP-binding P2X7 receptors (otherwise known as “non functionalreceptors”). The epitope and peptides including the epitope have beenfound to be useful for generating antibodies and immune complexes thatindicate the presence or absence or predisposition to a variety ofdiseases and conditions including carcinoma.

Thus in certain embodiments there is provided a peptide consisting ofthe sequence: SEQ ID NO: 2: KYYKENNVEKRTLIKVF

The peptide may consist of a sequence within the sequence of SEQ ID NO:2 (FIG. 2), in which case the peptide can be used as an immunogen togenerate an antibody that is capable of selectively binding to a nonATP-binding P2X7 (“non-functional”) receptor but not to an ATP-bindingP2X7 (“functional”) receptor. The term “non ATP-binding” and“non-functional” in relation to P2X7 may be used interchangeably in thespecification and claims. Similarly, the term “ATP-binding” and“functional” may be used interchangeably in the specification andclaims.

Generally a non ATP-binding receptor means a trimer or higher multimerformed from at least one P2X7 monomer, the trimer or higher multimerbeing at least substantially devoid of ATP binding activity. In certainembodiments, these trimers or higher multimers are substantially unableto form a pore in a cell membrane for ingress of calcium cations intothe cell cytoplasm.

Methods for screening peptides that can be used as an immunogen togenerate an antibody that is capable of selectively binding to a nonATP-binding P2X7 receptor but not to an ATP-binding P2X7 receptor aredisclosed herein. One example includes the use of erythrocytes in arosetting assay. In this assay an antibody that binds to functionalreceptors is used as a positive control in which rossettes are observed.A test antibody is determined not to bind to functional receptors if itfails to form rossettes. It is determined to bind to non functionalreceptors if it is observed to bind to a non functionalreceptor-expressing cell line, including those discussed herein.

In these embodiments, the peptide may have a length of 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16 or 17 residues.

In other embodiments, the peptide consists of a sequence of SEQ ID NO: 3(FIG. 3), said peptide being useful as an immunogen to generate anantibody that is capable of selectively binding to a non ATP-bindingP2X7 receptor but not to an ATP-binding P2X7 receptor.

The peptide may consist of a sequence within the sequence of SEQ ID NO:3, said peptide being useful as an immunogen to generate an antibodythat is capable of selectively binding to a non ATP-binding P2X7receptor but not to an ATP-binding P2X7 receptor. Examples of thesepeptides include those having a sequence described in Table 1 (numberingaccording to SEQ ID NO: 1):

TABLE 1 K281 to K297 Y298 to G314 T282 to Y298 Y299 to I315 T283 to Y299K300 to R316 N284 to K300 E301 to F317 V285 to E301 N302 to D318 S286 toN302 N303 to I319 L287 to N303 V304 to L320 Y288 to V304 E305 to V321P289 to E305 K306 to F322 G290 to K306 R307 to G323 Y291 to R307 T308 toT324 N292 to T308 L309 to G325 F293 to L309 I310 to G326 R294 to I310K311 to K327 Y295 to K311 V312 to F328 A296 to V312 F313 to D329 K297 toF313 (SEQ ID NO: 2)

The peptide shown in Table 1 may have a length of from 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16 or 17 residues.

In other embodiments there is provided an antibody or fragment thereofcapable of binding to a peptide described above.

The antibody may be produced by immunisation with a peptide as describedabove. An example of a suitable immunisation is described in Example 1below.

The antibody may also be produced by immunisation with a non ATP-bindingP2X7 receptor, such as a receptor having an amino acid sequence shown inSEQ ID NO: 1, or a fragment thereof including the amino acid sequencesSEQ ID NOS: 3 to 9 (shown in FIGS. 3 to 9).

In one embodiment, the non ATP-binding P2X7 receptor, monomer orfragment thereof used for the immunisation has Pro210 in cisisomerisation.

The antibody may bind to an epitope that includes one or more residuesof a peptide having a sequence of SEQ ID NO: 2. In certain embodiments,the antibody binds to K297 or Y298 or Y299 or K300 or E301 or N302 orN303 or V304 or E305 or K306 or R307 or T308 or L309 or I310 or K311 orV312 or F313

The epitope may include a sequence of residues of a peptide having asequence of SEQ ID NO: 2. In certain embodiments, the antibody binds toa sequence including K297 and Y298, or Y298 and Y299, or Y299 and K300,or K300 and E301, or E301 and N302, or N302 and N303, or N303 and V304,or V304 and E305, or E305 and K306, or K306 and R307, or R307 and T308,or T308 and L309, or L309 and I310, or I310 and K311, or K311 and V312,or V312 and F313.

The antibody that binds to one or more of the above residues may alsobind to one or more residues of the P2X7 receptor extra cellular domainthat are located outside of the region defined by SEQ ID NO:3. Forexample, the one or more residues located outside of the region definedby SEQ ID NO:3 may be located in a sequence of amino acid residues of anATP-binding P2X7 receptor that defines the ATP binding site of theATP-binding P2X7 receptor.

In one embodiment, the one or more residues located outside the sequenceof SEQ ID NO:3 may be located in the sequence of SEQ ID NOS: 10 to 12(FIGS. 10 to 12 respectively).

The antibody may be a whole antibody of any isotype. The antibody may beone obtained from monoclonal or polyclonal antisera. The antibody may beproduced by hybridoma, or by recombinant expression.

The antibody may be chimeric, i.e. one containing human variable domainsand non human constant domains. Alternatively, it may be humanized, i.eone formed by grafting non human CDRs onto a human antibody framework.Still further, the antibody may be fully human.

The antibodies of the invention may be modified with respect to effectorfunction, so as to enhance, e.g., the effectiveness of the antibody intreating cancer. For example, cysteine residue(s) can be introduced intothe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated can have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). Homodimericantibodies with enhanced anti-tumor activity can also be prepared usingheterobifunctional crosslinkers. Alternatively, an antibody can beengineered that has dual Fc regions and can thereby have enhancedcomplement lysis and ADCC capabilities.

Where the antibody is an antibody fragment, the antibody fragment isselected from the group consisting of a dAb, Fab, Fd, Fv, F(ab′)2, scFvand CDR.

The antibody or fragment may be provided on a solid phase such as abead, surface or tissue culture vessel.

The antibody or fragment thereof may be provided with a label fordetection of binding of the antibody or fragment thereof to antigen.

The antibodies and fragments may be labelled for use in medical imaging.Such methods involve chemical attachment of a labelling or imagingagent, such as a radioisotope, which include 67Cu, 90Y, 124I, 125I,131I, 186Re, 188Re, 211At, 212 Bi, administration of the labelledantibody or fragment to a subject in an acceptable carrier, and imagingthe labelled antibody or fragment in vivo at the target site.Radio-labelled antibodies or fragments thereof may be particularlyuseful in in vivo imaging of cancers described herein.

The antibodies can be purified by methods known to the skilled artisan.Purification methods include, among other, selective precipitation,liquid chromatography, HPLC, electrophoresis, chromatofocusing, andvarious affinity techniques.

In some embodiments, the antibodies disclosed herein may also includemultimeric forms of antibodies. For example, antibodies of the inventionmay take the form of antibody dimers, trimers, or higher-order multimersof monomeric immunoglobulin molecules.

Crosslinking of antibodies can be done through various methods known inthe art. For example, crosslinking of antibodies may be accomplishedthrough natural aggregation of antibodies, through chemical orrecombinant linking techniques or other methods known in the art. Forexample, purified antibody preparations can spontaneously form proteinaggregates containing antibody homodimers, and other higher-orderantibody multimers. In a specific embodiment, crosslinking of antibodiesby using a second antibody to bind to the antibodies of interest can beused to form a homodimer. The crosslinker antibody can be derived from adifferent animal compared to the antibody of interest. For example, agoat anti-mouse antibody (Fab specific) may be added to a mousemonoclonal antibody to form a homodimer. This bivalent crosslinkerantibody recognizes the Fab or Fc region of the two antibodies ofinterest forming a homodimer.

Alternatively, antibody homodimers may be formed through chemicallinkage techniques known in the art. Chemical crosslinkers can be homoor heterobifunctional and will covalently bind with two antibodiesforming a homodimer. In some embodiments, it is desirable that thechemical crosslinker not interact with the antigen-binding region of theantibody as this may affect antibody function. As will be appreciated bythose skilled in the art, antibodies can be crosslinked at the Fabregion.

In one embodiment there is provided an immune complex formed from thebinding of an antibody or fragment thereof as described above to a nonATP-binding (i.e. non-functional) P2X7 receptor, monomer or fragmentthereof. In one embodiment there is provided an immune complex formedfrom the binding of an antibody or fragment thereof to a peptidedescribed above.

Generally an immune complex otherwise known as an antigen-antibodycomplex is a product that is formed from the binding of an antibody viaan antibody binding site to an epitope on an antigen against which theantibody is raised. The complex may or may not consist of more than oneantibody.

The immune complex is particularly important as detection of this invitro or in vivo is indicative of presence of, or predisposition to adisease or condition including preneoplasia and neoplasia. Thesedetection methods are described in more detail below.

The non-ATP binding P2X7 receptor, monomer or fragment thereof includedin the immune complex may have Pro210 in cis isomerisation.

The non-ATP binding P2X7 receptor, monomer or fragment thereof includedin the immune complex may have an amino acid sequence of any one of SEQID NOS: 4 to 9 or fragments thereof.

The non-ATP binding P2X7 receptor, monomer or fragment thereof includedin the immune complex may have a molecular weight in the range of fromabout 15 to 80 kDa.

The non-ATP binding P2X7 receptor, monomer or fragment thereof includedin the immune complex may lack a transmembrane domain.

The immune complex may be formed by binding a non-ATP binding P2X7receptor, monomer or fragment thereof located on a cell surfacemembrane, in a cytoplasm, in a nucleus or in extra-cellular fluid. Theextra-cellular fluid may be blood, plasma, serum, lymph, urine, semen,saliva, sputum, ascites, faeces, uterine and vaginal secretions, bile,amniotic fluid, cerebrospinal fluid and organ and tissue flushings.

The antibody or antibody fragment included in the immune complex may beattached to a solid phase, such as a bead or a plate, so that the immunecomplex is attached to a solid phase when formed. Alternatively, thenon-ATP binding P2X7 receptor, monomer or fragment thereof included inthe immune complex may be attached to a solid phase.

The antibody may be labelled for detection of formation of the immunecomplex.

The immune complex may further include an antibody or fragment thereof,such as a capture antibody for capture of the immune complex. Thefurther antibody or fragment thereof may bind to the anti P2X7 receptorantibody. Also, the further antibody or fragment thereof may bind to thereceptor or fragment thereof.

The further antibody or fragment thereof may be bound to a solid phasesuch as a phase described above.

The further antibody may be labelled for detection of formation of theimmune complex. Examples of labels include fluorophores, dyes, isotopesetc.

In certain embodiments there is provided a method for determiningwhether a cell, tissue or extra cellular body fluid includes a nonATP-binding P2X7 receptor, monomer or fragment thereof including:

-   -   contacting a cell, tissue or extra cellular body fluid with an        antibody or fragment thereof in conditions for forming an immune        complex as described above, and    -   detecting whether an immune complex has been formed,

wherein detection of an immune complex determines that a cell, tissue orextra-cellular body fluid includes a non ATP-binding P2X7 receptor,monomer or fragment thereof.

In other embodiments there is provided a use of an antibody or fragmentthereof described above in the manufacture of means for determiningwhether a cell, tissue or extra-cellular body fluid contains a P2X7receptor, monomer or fragment thereof.

In other embodiments there is provided a method for determining whethera cell, tissue or extra-cellular body fluid contains an antibody againsta non ATP-binding P2X7 receptor, monomer or fragment thereof including:

-   -   contacting a cell, a tissue or an extra-cellular body fluid with        a peptide as described above in conditions for forming an immune        complex between the peptide and an antibody in the cell, tissue        or extra-cellular body fluid, and    -   detecting whether an immune complex has been formed,

wherein detection of an immune complex determines that a cell, tissue orextra-cellular body fluid contains an antibody against a non ATP-bindingP2X7 receptor, monomer or fragment thereof.

In other embodiments there is provided a use of a peptide describedabove in the manufacture of means for determining whether a cell, tissueor extra-cellular body fluid contains an antibody against a nonATP-binding P2X7 receptor, monomer or fragment thereof.

The presence of a given protein, or level of expression of a givenprotein in a host cell, tissue or extra-cellular body fluid can bedetected by any number of assays. Examples include immunoassays,chromatography and mass spectrometry.

Immunoassays, i.e. assays involving an element of the immune system areparticularly preferred. These assays may generally be classified intoone of:

(i) assays in which purified antigen is used to detect an antibody inhost serum. For example, purified antigen is bound to solid phase byadsorption or indirectly through another molecule and host serum isapplied followed by another antibody for detecting presence or absenceof host antibody;

(ii) assays in which purified antigen is used to detect immune cells,such as T and B lymphocytes. For example, peripheral white cells arepurified from a host and cultured with purified antigen. The presence orabsence of one or factors indicating immunity are then detected. Otherexamples include assays that measure cell proliferation (lymphocyteproliferation or transformation assays) following exposure to purifiedantigen, and assays that measure cell death (including apoptosis)following exposure to purified antigen;

(iii) assays in which purified antibody specific for antigen is used todetect antigen in the host. For example, purified antibody is bound tosolid phase, host tissue is then applied followed by another antibodyspecific for the antigen to be detected. There are many examples of thisapproach including ELISA, RIA;

(iv) assays in which a purified anti-idiotypic antibody is used todetect host antibody. For example, anti-idiotypic antibody is adsorbedto solid phase, host serum is added and anti-Fc antibody is added tobind to any host antibodies having been bound by the anti-idiotypicantibody.

The immunoassays can be applied in vitro or in vivo.

In one embodiment, the disease is typically a cancer such as carcinoma,sarcoma, lymphoma, or leukemia. Carcinomas that may be detected include,but not limited to, prostate, breast, skin, lung, cervix, uterus,stomach, oesophagus, bladder, and colon cancers.

Whilst any body fluid can be used to detect any of these diseases, somebody fluids may be more appropriate than others to detect certaindiseases, for example urine may be more appropriate to detect prostatecancer and blood for detecting blood cancers such as lymphoma.

In another embodiment there is provided a method for determining whetheran individual has a cancer including the steps of:

-   -   contacting a cell, a tissue or an extra-cellular body fluid with        a peptide as described above in conditions for forming an immune        complex between the peptide and an antibody in the cell, tissue        or extra-cellular body fluid; or    -   contacting a cell, tissue or extra cellular body fluid with an        antibody or fragment thereof in conditions for forming an immune        complex as described above, and    -   detecting whether an immune complex has been formed,

wherein detection of an immune complex determines that an individual hasa cancer.

In a further embodiment there is provided use of anti purinergic (P2X)receptor antibody or fragment thereof as described above, or a peptideas described above for determining whether an individual has a cancer.

In certain embodiments, cancer is selected from the group consisting ofprostate cancer, invasive breast cancer, melanoma, adenocarcinoma of thebowel, serous ovarian cancer, squamous cell cancer of the cervix,endometrial cancer, small cell lung cancer, hepatocellular carcinoma,transitional cell carcinoma of the bladder, gastrointestinal stromaltumour, endometrial stromal tumour, pituitary cancer, mesothelioma,Hodgkin's lymphoma and thyroid papillary.

In yet further embodiments there is provided a kit or composition fordetermining whether a cell, tissue or extra-cellular body fluid containsa non ATP-binding P2X7 receptor, monomer or fragment thereof, or anantibody against same including:

-   -   a peptide as described above; and/or    -   an antibody or fragment thereof as described above; and/or    -   non ATP-binding P2X7 receptor, monomer or fragment thereof; and        optionally    -   a further antibody for binding to the peptide, antibody or        fragment thereof or the non ATP-binding P2X7 receptor, monomer        or fragment thereof;    -   written instructions for use of the kit in a method described        above.

Kits are provided which contain the necessary reagents to carry out theassays of the present invention. The kit may include one or morecompartments, each to receive one or more containers such as: (a) afirst container comprising one of the components of the presentinvention described above; and (b) one or more other containerscomprising one or more of the following: wash reagents, reagents capableof detecting presence of the antibody or peptide.

The containers allow one to efficiently transfer reagents from onecompartment to another compartment such that the samples and reagentsare not cross-contaminated, and the agents or solutions of eachcontainer can be added in a quantitative fashion from one compartment toanother.

The kit typically contains containers which may be formed from a varietyof materials such as glass or plastic, and can include for example,bottles, vials, syringes, and test tubes. A label typically accompaniesthe kit, and includes any writing or recorded material, which may beelectronic or computer readable form (e.g., disk, optical disc, or tape)providing instructions or other information for used of the contents ofthe kit. The label indicates that the formulation is used for diagnosingor treating the disorder of choice.

One skilled in the art will readily recognize that the disclosedantibodies and peptides of the present invention can be readilyincorporated into one of the established kit formats which are wellknown in the art.

In other embodiments there is provided a pharmaceutical compositionincluding an antibody or fragment thereof as described above, or apeptide as described above, together with a pharmaceutically acceptablecarrier, diluent or excipient.

In the preparation of the pharmaceutical compositions comprising theantibodies or peptides described in the teachings herein, a variety ofvehicles and excipients and routes of administration may be used, aswill be apparent to the skilled artisan. Representative formulationtechnology is taught in, inter alia, Remington: The Science and Practiceof Pharmacy, 19th Ed., Mack Publishing Co., Easton, Pa. (1995) andHandbook of Pharmaceutical Excipients, 3rd Ed, Kibbe, A. H. ed.,Washington D.C., American Pharmaceutical Association (2000); herebyincorporated by reference in their entirety.

The pharmaceutical compositions will generally comprise apharmaceutically acceptable carrier and a pharmacologically effectiveamount of the antibodies or peptides, or mixture of antibodies ormixture of peptides, or suitable salts thereof.

The pharmaceutical composition may be formulated as powders, granules,solutions, suspensions, aerosols, solids, pills, tablets, capsules,gels, topical creams, suppositories, transdermal patches, and otherformulations known in the art.

For the purposes described herein, pharmaceutically acceptable salts ofthe antibodies and peptides is intended to include any art recognizedpharmaceutically acceptable salts including organic and inorganic acidsand/or bases. Examples of salts include sodium, potassium, lithium,ammonium, calcium, as well as primary, secondary, and tertiary amines,esters of lower hydrocarbons, such as methyl, ethyl, and propyl. Othersalts include organic acids, such as acetic acid, propionic acid,pyruvic acid, maleic acid, succinic acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, salicylic acid, etc.

As used herein, “pharmaceutically acceptable carrier” comprises anystandard pharmaceutically accepted carriers known to those of ordinaryskill in the art in formulating pharmaceutical compositions. Thus, theantibodies or peptides, by themselves, such as being present aspharmaceutically acceptable salts, or as conjugates, may be prepared asformulations in pharmaceutically acceptable diluents; for example,saline, phosphate buffer saline (PBS), aqueous ethanol, or solutions ofglucose, mannitol, dextran, propylene glycol, oils (e.g., vegetableoils, animal oils, synthetic oils, etc.), microcrystalline cellulose,carboxymethyl cellulose, hydroxylpropyl methyl cellulose, magnesiumstearate, calcium phosphate, gelatin, polysorbate 80 or as solidformulations in appropriate excipients.

The pharmaceutical compositions will often further comprise one or morebuffers (e.g., neutral buffered saline or phosphate buffered saline),carbohydrates (e.g., glucose, sucrose or dextrans), mannitol, proteins,polypeptides or amino acids such as glycine, antioxidants (e.g.,ascorbic acid, sodium metabisulfite, butylated hydroxytoluene, butylatedhydroxyanisole, etc.), bacteriostats, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminium hydroxide), solutes that renderthe formulation isotonic, hypotonic or weakly hypertonic with the bloodof a recipient, suspending agents, thickening agents and/orpreservatives. Alternatively, compositions of the present invention maybe formulated as a lyophilizate.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the compositions of this invention, the type ofcarrier will typically vary depending on the mode of administration.Antibody and peptide compositions may be formulated for any appropriatemanner of administration, including for example, oral, nasal, mucosal,intravenous, intraperitoneal, intradermal, subcutaneous, andintramuscular administration.

For parenteral administration, the compositions can be administered asinjectable dosages of a solution or suspension of the substance in aphysiologically acceptable diluent with a pharmaceutical carrier thatcan be a sterile liquid such as sterile pyrogen free water, oils,saline, glycerol, polyethylene glycol or ethanol. Additionally,auxiliary substances, such as wetting or emulsifying agents,surfactants, pH buffering substances and the like can be present incompositions.

Other components of pharmaceutical compositions are those of petroleum,animal, vegetable, or synthetic origin, for example, non-aqueoussolutions of peanut oil, soybean oil, corn oil, cottonseed oil, ethyloleate, and isopropyl myristate. Antibodies and peptides can beadministered in the form of a depot injection or implant preparationwhich can be formulated in such a manner as to permit a sustainedrelease of the active ingredient. An exemplary composition comprisesantibody at 5 mg/ml, formulated in aqueous buffer consisting of 50 mML-histidine, 150 mM NaCl, adjusted to pH 6.0 with HCl.

Typically, the compositions are prepared as injectables, either asliquid solutions or suspensions; solid or powder forms suitable forreconstitution with suitable vehicles, including by way example and notlimitation, sterile pyrogen free water, saline, buffered solutions,dextrose solution, etc., prior to injection. The preparation also can beemulsified or encapsulated in liposomes or micro particles such aspolylactide, polyglycolide, or copolymers.

The pharmaceutical compositions described herein may be presented inunit-dose or multi-dose containers, such as sealed ampoules or vials.Such containers are typically sealed in such a way to preserve thesterility and stability of the formulation until use. In general,formulations may be stored as suspensions, solutions or emulsions inoily or aqueous vehicles, as indicated above.

Alternatively, a pharmaceutical composition may be stored in afreeze-dried condition requiring only the addition of a sterile liquidcarrier immediately prior to use.

In related embodiments there is provided a method of treatment of adisease characterised by the expression of a non ATP-binding P2X7receptor, monomer or fragment thereof including the step of providing anantibody or fragment thereof as described above, or a peptide asdescribed above to an individual requiring said treatment.

Methods of immunotargeting cancer cells using antibodies or antibodyfragments are well known in the art. U.S. Pat. No. 6,306,393 describesthe use of anti-CD22 antibodies in the immunotherapy of B-cellmalignancies, and U.S. Pat. No. 6,329,503 describes immunotargeting ofcells that express serpentine transmembrane antigens. Antibodiesdescribed herein (including humanized or human monoclonal antibodies orfragments or other modifications thereof, optionally conjugated tocytotoxic agents) can be introduced into a patient such that theantibody binds to cancer cells and mediates the destruction of the cellsand the tumor and/or inhibits the growth of the cells or the tumor.

Without intending to limit the disclosure, mechanisms by which suchantibodies can exert a therapeutic effect may includecomplement-mediated cytolysis, antibody-dependent cellular cytotoxicity(ADCC)1 modulating the physiologic function of the tumor antigen,inhibiting binding or signal transduction pathways, modulating tumorcell differentiation, altering tumor angiogenesis factor profiles,modulating the secretion of immune stimulating or tumor suppressingcytokines and growth factors, modulating cellular adhesion, and/or byinducing apoptosis.

The antibodies can also be conjugated to toxic or therapeutic agents,such as radioligands or cytosolic toxins, and may also be usedtherapeutically to deliver the toxic or therapeutic agent directly totumor cells.

By “treatment” herein is meant therapeutic or prophylactic treatment, ora suppressive measure for the disease, disorder or undesirablecondition. Treatment encompasses administration of the subjectantibodies in an appropriate form prior to the onset of disease symptomsand/or after clinical manifestations, or other manifestations, of thedisease to reduce disease severity, halt disease progression, oreliminate the disease. Prevention of the disease includes prolonging ordelaying the onset of symptoms of the disorder or disease, preferably ina subject with increased susceptibility to the disease.

The therapeutic preparations can use nonmodified antibodies orantibodies conjugated with a therapeutic compound, such as a toxin orcytotoxic molecule, depending on the functionality of the antibody.Generally, when nonmodified antibodies are used, they will typicallyhave a functional Fc region. By “functional Fc region” herein is meant aminimal sequence for effecting the biological function of Fc, such asbinding to Fc receptors, particularly FcγR (e.g., FcγR1, RIIA, RIIB,RIIIA, RIIIB).

Without being bound by theory, it is believed that the Fc region mayaffect the effectiveness of anti tumor monoclonal antibodies by bindingto Fc receptors immune effector cells and modulating cell mediatedcytotoxicity, endocytosis, phagocytosis, release of inflammatorycytokines, complement mediate cytotoxicity, and antigen presentation. Inthis regard, polyclonal antibodies, or mixtures of monoclonals will beadvantageous because they will bind to different epitopes and thus havea higher density of Fc on the cell surface as compared to when a singlemonoclonal antibody is used. Of course, to enhance their effectivenessin depleting targeted cells, or where nonmodified antibodies are nottherapeutically effective, antibodies conjugated to toxins or cytotoxicagents may be used.

The antibody compositions may be used either alone or in combinationwith other therapeutic agents to increase efficacy of traditionaltreatments or to target abnormal cells not targeted by the antibodies.Combining the antibody therapy method with a chemotherapeutic, radiationor surgical regimen may be preferred in patients that have not receivedchemotherapeutic treatment, whereas treatment with the antibody therapymay be indicated for patients who have received one or morechemotherapies. Additionally, antibody therapy can also enable the useof reduced dosages of concomitant chemotherapy, particularly in patientsthat do not tolerate the toxicity of the chemotherapeutic agent verywell. Furthermore, treatment of cancer patients with the antibody withtumors resistant to chemotherapeutic agents might induce sensitivity andresponsiveness to these agents in combination.

In one aspect, the antibodies are used adjunctively with therapeuticcytotoxic agents, including, by way of example and not limitation,busulfan, thioguanine, idarubicin, cytosine arabinoside,6-mercaptopurine, doxorubicin, daunorubicin, etoposide, and hydroxyurea.Other agents useful as adjuncts to antibody therapy are compoundsdirected specifically to the abnormal cellular molecule found in thedisease state. These agents will be disease specific. For example, fortreating chronic myeloid leukemia arising from BCR-ABL activity, oneclass of useful compounds are inhibitors of abl kinase activity, such asImatinib, an inhibitor of bcr-abl kinase, and antisense oligonucleotidesagainst bcr (e.g., Oblimersen). Other agents include, among others,interferon-alpha, humanized anti-CD52, deacetylase inhibitor FR901228(depsipeptide), and the like.

The amount of the compositions needed for achieving a therapeutic effectwill be determined empirically in accordance with conventionalprocedures for the particular purpose. Generally, for administering thecompositions ex vivo or in vivo for therapeutic purposes, thecompositions are given at a pharmacologically effective dose. By“pharmacologically effective amount” or “pharmacologically effectivedose” is an amount sufficient to produce the desired physiologicaleffect or amount capable of achieving the desired result, particularlyfor treating or retreating the disorder or disease condition, includingreducing or eliminating one or more symptoms or manifestations of thedisorder or disease.

As an illustration, administration of antibodies to a patient sufferingfrom prostate cancer provides a therapeutic benefit not only when theunderlying disease is eradicated or ameliorated, but also when thepatient reports a decrease in the severity or duration of the symptomsassociated with the disease. Therapeutic benefit also includes haltingor slowing the progression of the underlying disease or disorder,regardless of whether improvement is realized.

The amount administered to the host will vary depending upon what isbeing administered, the purpose of the administration, such asprophylaxis or therapy, the state of the host, the manner ofadministration, the number of administrations, interval betweenadministrations, and the like. These can be determined empirically bythose skilled in the art and may be adjusted for the extent of thetherapeutic response. Factors to consider in determining an appropriatedose include, but is not limited to, size and weight of the subject, theage and sex of the subject, the severity of the symptom, the stage ofthe disease, method of delivery of the agent, half-life of the agents,and efficacy of the agents. Stage of the disease to consider includeswhether the disease is acute or chronic, relapsing or remitting phase,and the progressiveness of the disease. Determining the dosages andtimes of administration for a therapeutically effective amount are wellwithin the skill of the ordinary person in the art.

For any compositions of the present disclosure, the therapeuticallyeffective dose is readily determined by methods well known in the art.For example, an initial effective dose can be estimated from cellculture or other in vitro assays. For example, Sliwkowsky, M X et al.,Semin. Oncol. 26. suppl. 12) 60-70 (1999) describes in vitromeasurements of antibody dependent cellular cytoxicity. A dose can thenbe formulated in animal models to generate a circulating concentrationor tissue concentration, including that of the IC50 as determined by thecell culture assays.

In addition, the toxicity and therapeutic efficacy are generallydetermined by cell culture assays and/or experimental animals, typicallyby determining a LD50 (lethal dose to 50% of the test population) andED50 (therapeutically effectiveness in 50% of the test population). Thedose ratio of toxicity and therapeutic effectiveness is the therapeuticindex. Preferred are compositions, individually or in combination,exhibiting high therapeutic indices. Determination of the effectiveamount is well within the skill of those in the art, particularly giventhe detailed disclosure provided herein. Guidance is also found instandard reference works, for example Fingl and Woodbury, GeneralPrinciples In: The Pharmaceutical Basis of Therapeutics pp. 1-46 (1975),and the references cited therein.

To achieve an initial tolerizing dose, consideration is given to thepossibility that the antibodies may be immunogenic in humans and innon-human primates. The immune response may be biologically significantand may impair the therapeutic efficacy of the antibody even if theantibody is partly or chiefly comprised of human immunoglobulinsequences such as, for example, in the case of a chimeric or humanizedantibody. Within certain embodiments, an initial high dose of antibodyis administered such that a degree of immunological tolerance to thetherapeutic antibody is established.

The tolerizing dose is sufficient to prevent or reduce the induction ofan antibody response to repeat administration of the committedprogenitor cell specific antibody.

Preferred ranges for the tolerizing dose are between 10 mg/kg bodyweight to 50 mg/kg body weight, inclusive. More preferred ranges for thetolerizing dose are between 20 and 40 mg/kg, inclusive. Still morepreferred ranges for the tolerizing dose are between 20 and 25 mg/kg,inclusive.

Within these therapeutic regimens, the therapeutically effective dose ofantibodies is preferably administered in the range of 0.1 to 10 mg/kgbody weight, inclusive. More preferred second therapeutically effectivedoses are in the range of 0.2 to 5 mg/kg body weight, inclusive. Stillmore preferred therapeutically effective doses are in the range of 0.5to 2 mg/kg, inclusive. Within alternative embodiments, the subsequenttherapeutic dose or doses may be in the same or different formulation asthe tolerizing dose and/or may be administered by the same or differentroute as the tolerizing dose.

For the purposes of this invention, the methods of administration arechosen depending on the condition being treated, the form of the subjectantibodies, and the pharmaceutical composition.

Administration of the antibody compositions can be done in a variety ofways, including, but not limited to, continuously, subcutaneously,intravenously, orally, topically, transdermal, intraperitoneal,intramuscularly, and intravesically. For example, microparticle,microsphere, and microencapsulate formulations are useful for oral,intramuscular, or subcutaneous administrations. Liposomes andnanoparticles are additionally suitable for intravenous administrations.Administration of the pharmaceutical compositions may be through asingle route or concurrently by several routes. For instance,intraperitoneal administration can be accompanied by intravenousinjections. Preferably the therapeutic doses are administeredintravenously, intraperitonealy, intramuscularly, or subcutaneously.

The compositions may be administered once or several times. In someembodiments, the compositions may be administered once per day, a few orseveral times per day, or even multiple times per day, depending upon,among other things, the indication being treated and the judgement ofthe prescribing physician.

Administration of the compositions may also be achieved throughsustained release or long-term delivery methods, which are well known tothose skilled in the art. By “sustained release or” “long term release”as used herein is meant that the delivery system administers apharmaceutically therapeutic amount of subject compounds for more than aday, preferably more than a week, and most preferable at least about 30days to 60 days, or longer. Long term release systems may compriseimplantable solids or gels containing the antibodies, such asbiodegradable polymers described above; pumps, including peristalticpumps and fluorocarbon propellant pumps; osmotic and mini-osmotic pumps;and the like.

The method of the invention contemplates the administration of singlemonoclonal antibodies and any antibody that recognizes the particularantigens recognized by these antibodies, as well as combinations, ofdifferent mAbs. Two or more monoclonal antibodies may provide animproved effect compared to a single antibody. Alternatively, acombination of an antibody with an antibody that binds a differentantigen may provide an improved effect compared to a single antibody.Such mAb cocktails may have certain advantages inasmuch as they containmAbs, which exploit different effector mechanisms or combine directlycytotoxic mAbs with mAbs that rely on immune effector functionality.Such mAbs in combination may exhibit synergistic therapeutic effects.

It will be understood that the invention disclosed and defined in thisspecification extends to all alternative combinations of two or more ofthe individual features mentioned or evident from the text or drawings.All of these different combinations constitute various alternativeaspects of the invention.

The following protocols are provided as non-limiting examples for thepurpose of illustrating the invention.

EXAMPLES Example 1

Anti-nonfunctional P2X₇ antibody was raised against the peptideKYYKENNVEKRTLIKVF (SEQ ID NO: 2) orLys-Tyr-Tyr-Lys-Glu-Asn-Asn-Val-Glu-Lys-Arg-Thr-Leu-Ile-Lys-Val-Phe,representing amino acids 297-313 of the human P2X₇ protein. The peptidewas synthesized by solid phase chemistry (Mimotopes Pty Ltd, Melbourne,Australia) to high purity (>95%) as judged by mass spectroscopy. AC-terminal Cys (C) residue was attached to the sequence and to this wasattached the cross-linker MCS (6-Maleimido-Caproic AcidN-Hydroxysuccinimide Ester) for conjugating the peptide to carrierproteins including separately Diphtheria toxoid, bovine serum albuminand ovalbumin by Mimotopes.

Example 2

Female New Zealand White rabbits, aged between 10-12 weeks, wereimmunized according to the following schedule.

On day one, each rabbit received 200 ug of P2X₇ epitope conjugated todiphtheria toxoid (total antigen weight ˜500 ug of conjugated epitope).This conjugated peptide was supplied by Mimotopes Pty Ltd as a stablesolution.

The epitope conjugate was diluted in sterile PBS to a concentration of500 ug per 0.8 ml PBS. To this was added 0.1 ml of DEAE/Dextran/QUILA™solution (2.5 mg QUILA™ plus 25 mg DEAE/Dextran per mL of PBS) and 1.2mL of Montanide 15A50V. This solution was emulsified using glass luerlocsyringes and a narrow bore luerloc coupling.

Animals were each injected with 2 ml of the epitope adjuvant emulsion atmultiple subcutaneous and intra-muscular sites.

The above was repeated at 6-weeks and 9-weeks post initial injections.At week 10, the rabbits were injected intravenously with 1 ml of sterilePBS containing 50 ug of DT-conjugated P2X₇ epitope. Four days later, therabbits were bled out and the serum containing the antibody was storedfor future analysis and use in immunoassays.

Example 3

The procedure in Example 2 was used except that mice were injected with20 ug of P2X₇ epitope (˜50 ug of DT-conjugate) in 0.2 mL of epitopeadjuvant emulsion.

Four days after the intravenous injection, antibody titers were measuredin mouse blood and the highest titer mice were selected as spleen donorsfor hybridoma fusions.

Example 4

Mice selected above were used as spleen cell donors and these cells werefused with mouse SP20 myeloma cells to form hybridoma cell linesaccording to the 96-well plate format modification of the originalprotocol described by Kohler and Milstein.

Cell lines were selected for stability and production of the specificantibody to particular P2X₇ epitopes.

Example 5

A monoclonal antibody with suitably high affinity for the target epitopewas selected for the IHC study. The binding characteristics of theantibody was tested by measuring the interaction with the target epitopeon a Biacore instrument. A total of 550 resonance units of binding wasachieved in the 60 second loading time showing slow on rate. A very slowoff rate was apparent following cessation of loading with no measurablediminution of binding over the subsequent 10 minutes.

Example 6

Binding of the monoclonal antibody to fixed and permeabilized C11STHcells expressing the non-functional receptor was performed on a flowcytometer (Becton-Dickenson) using Alexa-488 label. The mean value was90.8 compared with negative controls of 1.9. Binding of the monoclonalantibody to receptors on the surface of unfixed live cells was alsoassessed. The mean value was 5.8 compared with 0.34 for the negativecontrol.

Example 7

For fixed cells, standard fluorescent antibody staining and confocalmicroscopy was used as follows. Fixed cells on poly-L-lysine-coatedglass coverslips in 48-well plates were incubated with 20% normal horseserum in phosphate buffered saline pH7.5 (PBS) for 20 minutes, washedwith PBS for 5 minutes then incubated with primary antibody for 30 min,washed with PBS for 5 minutes and finally labeled with fluorescentlabeled secondary antibody (Jackson Immunologics) for 30 minutes. Cellswere then washed twice (2×5 minutes) with PBS before mounting thecoverslips on slides in 50% glycerol in PBS. Cells were visualized witha Leica TCS NT UV laser confocal microscope system with the pinhole setat 1.0. Murine isotype control antibodies were used routinely asnegative controls and showed no staining. Western blots of HEK293 cellstransfected with P2X7 revealed a single band at approximately 75 kDathat was absent in non-transfected cells and in samples of homogenatespre-treated with the epitope. Cell protein extracts (30 ug) togetherwith molecular weight markers were fractionated on sodium dodecylsulfate polyacrylamide gel (8-16%) (Novex). Proteins wereelectro-blotted on Immobilon-P membrane (Millipore). Western blots weredeveloped using the ECL-chemiluminescence system (Amersham).

Example 8

A total of 25 different cases of breast cancers, 25 cases of skincancers and 25 cases of prostate cancers were examined byimmunohistochemistry. There was no cross-reactivity between thefunctional receptors present on the red blood cells and thenon-functional receptor antibody (data not shown).

Examples of cancer tissue including prostate cancer, breast cancer asrepresented by ductal carcinoma in situ and melanoma all stained fornon-functional P2X₇. The epithelium in normal tissues with no adjacenttumour was devoid of the receptor while all cancer cases stained for thereceptor.

Cancer cell lines derived from these and other tissues an other weresimilarly found to express the receptors. Examples tested and foundpositive include ADDP, PC3, LNCap, MCF7, MDA-MB-235, MDA-MB-431,NCIH460, NCIH69, NCIH596, DU145, ACHN, 786-O, Hep3B2, C11STH and BT474.Similar patterns of expression were seen on tumor xenografts of ovarianADDP and lung NCIH69 respectively. The non-functional P2X₇ receptorswere found on the plasma membrane with correspondingly little residualcytoplasmic receptor in the most advanced stages of the cancers. Incontrast, lower grade tumours exhibited a preponderance of cells inwhich a large proportion of the receptors remain intracellular. Such aprogressive transport of non-functioning apoptotic receptors to theplasma membrane over the course of the disease progression indicatesthat cancer cell lines should exhibit non-functional P2X₇ receptors onthe plasma membrane rather than being largely intracellular.

Normal epithelial tissue is devoid of the non-functional receptors whilecancer tissues are all labelled. Different breast cancers, includingboth invasive and in situ lobular and ductal carcinomas were examined.All types expressed non-functional receptors. Epithelial cells in theseand other normal, non-cancerous tissues such as bowel, bladder, ovarian,uterine, cervical, stomach and lung were found to be devoid ofnon-functional receptors.

Areas of morphologically normal tissue surrounding tumours in prostatealso expressed the receptors consistent with the field-effect in whichtumour cells influence surrounding normal epithelium in connected ductsthereby alerting these cells to the presence of a developing tumour. Inresponse, these cells begin deploying receptor, initially in an entirelyintracellular location but eventually on the plasma membrane.

In addition to the breast, skin and prostate cancer samples, otherexamples of epithelial cell cancers, all of which expressednon-functional P2X₇ included bowel adenocarcinoma, invasive ovariancancer, squamous cell carcinoma of the cervix, endometrial carcinoma ofthe uterus, small cell lung cancer, hepatocellular carcinoma,transitional cell carcinoma of the bladder and Barrett's mucosa withadenocarcinoma.

Human cancers of non-epithelial cell origin were also examined. All werefound to express the non-functional P2X₇ receptors. Examples ofmesenchymal cancers include gastrointestinal stromal tumour andendometrial stromal tumour. Equally, other tumours derived from thiscell type such as Ewing's sarcoma express the receptors as do braintumours such as oligodendrogliomas and astrocytomas, as do pituitarycarcinoma. Mesothelioma, a cancer derived from pleural cells, alsoexpress non functional receptors, as do solid tumours derived from bloodcells such as mantle cell lymphomas. Hodgkin's lymphoma and thyroidpapillary are examples.

Cross-reactivity between human and other mammalian cancers such asprostate, breast and skin from dogs and cats and melanoma in mice wasobserved using the same antibody to human receptor. These observationsadd weight to the conclusion that the cancer cell target is ubiquitous.

Example 9

There are indications that the receptor expression alters with tumourgrade providing the potential to differentiate between latent andaggressive forms of cancer. Certainly, very slow growing low gradeprostate cancers exhibit a pattern of receptor expression that is almostentirely intracellular, while cases of invasive prostate cancer exhibitmore plasma membrane and myoepithelial cell labelling together with asignificantly elevated receptor density.

Low grade and clinically unimportant prostate cancer can bedifferentiated from clinically important prostate cancer by theappearance of the surrounding areas of normal epithelium. Clinicallyimportant cancers include those that are likely to spread into theprostatic stroma and metastasize. Direct sampling of the tumour withtrans rectal ultrasound (TRUS) guided needle biopsies may detect aclinically significant tumour, thus making diagnosis straightforward. Incases in which the extant tumour is sampled in a region that is lowergrade (eg Gleason 3+3) with other areas (eg Gleason 3+4) missed,staining for the non-functional P2X₇ receptor shows that areas of normalmorphology also sampled in the prostate show intense levels of receptorexpression and myoepithelial stain, indicating the presence of tissuethat is metastasizing. Even cases in which the tumour is completelymissed, such as tumours confined to the apical lobe out of reach of theneedles, the presence and status of the tumour is readily detected asthe field-effect emanating from the tumour to surrounding epitheliumalerts the pathologist to the presence of the tumour and the likelygrade.

Example 10

Dysplastic tissue is difficult to diagnose and conditions such asBarrett's mucosa with dysplasia need to be monitored given thepropensity of the tissue to transform to adenocarcinoma. Thedifferentiation between dysplastic tissue that will remain benign andtissue that is at extreme risk of imminent transformation again centreson the presence of associated myoepithelial stain showing a dramaticup-regulation of non-functional P2X₇ receptor expression. Similarresults were observed for other dysplastic bowel conditions such ascolitis. In tissue samples of Barrett's with dysplasia there was nostaining indicating a benign condition while samples with intensemyoepithelial stain identified a subject that had an associatedadenocarcinoma.

Early cases of neoplastic transformation including cervicalintraepithelial neoplasia (CIN) grades 1-3 also showed increased levelsof receptor expression and cases such as ovarian serous borderline werecategorised as being benign (unstained) and those that were going totransform (heavy epithelial stain).

The above results indicate that an epitope that includes one or moreamino acids of the sequence KYYKENNVEKRTLIKVF (SEQ ID NO:2) hasexpression on a broad election of epithelial cancer tissues and cells.

The invention claimed is:
 1. A method of treatment of cancer in asubject in need thereof, the method comprising the step of administeringto the subject a therapeutically effective amount of a peptideconsisting of the sequence shown in SEQ ID NO: 3, or a peptideconsisting of a sequence within the sequence shown in SEQ ID NO: 3, saidpeptide being useful as an immunogen to generate an antibody that iscapable of selectively binding to a non ATP-binding P2X₇ receptor butnot to an ATP-binding P2X₇ receptor, and wherein the peptide includesthe sequence shown in SEQ ID NO:
 2. 2. The method according to claim 1,wherein the peptide consists of the sequence shown in SEQ ID NO:
 2. 3.The method according to claim 1, wherein the peptide consists of thesequence shown in SEQ ID NO:
 3. 4. The method according to claim 1,wherein the peptide is linked to a heterologous carrier for assistingwith generating the antibody.
 5. The method according to claim 4,wherein the carrier is selected from bovine serum albumin, ovalbumin ordiphtheria toxoid.
 6. The method according to claim 1, wherein thepeptide is administered in a pharmaceutical composition together with apharmaceutically acceptable carrier, diluent or excipient.
 7. A methodof treatment of cancer in a subject in need thereof, the methodcomprising the step of administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition including a peptideconsisting of the sequence as shown in SEQ ID NO: 3, or a peptideconsisting of a sequence within the sequence shown in SEQ ID NO: 3, saidpeptide being useful as an immunogen to generate an antibody that iscapable of selectively binding to a non ATP-binding P2X₇ receptor butnot to an ATP-binding P2X₇ receptor and wherein the composition furthercomprises an adjuvant for assisting with the generation of the antibody.8. The method according to claim 7, wherein the peptide consists of thesequence shown in SEQ ID NO:
 2. 9. The method according to claim 7,wherein the peptide consists of the sequence shown in SEQ ID NO:
 3. 10.The method according to claim 7, wherein the adjuvant is aluminiumhydroxide or DEAE-Dextran.
 11. The method according to claim 1, whereinthe cancer is selected from prostate, breast, skin, lung, cervix,uterus, ovarian, stomach, oesophagus, bladder, and colon cancers. 12.The method according to claim 7, wherein the cancer is selected fromprostate, breast, skin, lung, cervix, uterus, ovarian, stomach,oesophagus, bladder, and colon cancers.